Method for detection of binding affinities

ABSTRACT

A method for the detection of binding affinities comprises providing a device having a planar waveguide ( 2 ) arranged on a substrate ( 3 ) and an optical coupler ( 4 ). Coherent light ( 1 ) of a predetermined wavelength is coupled into the planar waveguide ( 2 ) such that the coherent light propagates along the planar waveguide ( 2 ), with an evanescent field ( 6 ) of the coherent light propagating along an outer surface ( 5 ) of the planar waveguide ( 2 ). Target samples ( 8 ) attached to binding sites ( 7 ) are arranged along a plurality of predetermined lines ( 9 ) on the outer surface ( 5 ) of the planar waveguide ( 2 ). At a predetermined detection location, light of the evanescent field which is scattered by target samples ( 8 ) bound to binding sites ( 7 ) arranged along the predetermined lines ( 9 ) is detected. The light scattered by the target samples ( 8 ) bound to the binding sites ( 7 ) has, at the predetermined detection location, a difference in optical path length which is an integer multiple of the predetermined wavelength of the light.

CROSS REFERENCE TO RELATED APPLICATIONS

This disclosure is a continuation application of U.S. patent applicationSer. No. 14/372,707 filed on Jul. 16, 2014, which is a 35 U.S.C. § 371national stage entry of PCT/EP2013/050825, which has an internationalfiling date of Jan. 17, 2013, which claims priority to European PatentApplication No. 12151436.8 filed on Jan. 17, 2012. The disclosures ofthe patent applications referenced above are herein incorporated byreference in their entireties.

FIELD

The present invention relates to a device for use in the detection ofbinding affinities as well as a system and a method for the detection ofbinding affinities in accordance with the respective independent claim.

BACKGROUND

Such devices are used, for example, as biosensors in a large variety ofapplications. One particular application is the detection or monitoringof binding affinities or processes. For example, with the aid of suchbiosensors various assays detecting the binding of target samples tobinding sites can be performed. Typically, large numbers of such assaysare performed on a biosensor at spots which are arranged in atwo-dimensional microarray on the surface of the biosensor. The use ofmicroarrays provides a tool for the simultaneous detection of thebinding affinities or processes of different target samples inhigh-throughput screenings, wherein large amounts of target samples likemolecules, proteins or DNA can be analysed quickly. For detecting theaffinities of target samples to bind to specific binding sites (e.g. theaffinities of target molecules to bind to different capture molecules),a large number of binding sites is immobilised on the surface of thebiosensor at spots which can be applied, for instance, by ink-jetspotting. Each spot forms an individual measurement zone for apredetermined type of capture molecules. The affinity of a target sampleto a specific type of capture molecules is detected and is used toprovide information on the binding affinity of the target sample.

A known technique for detecting binding affinities of target samplesuses labels which are capable of emitting fluorescent light uponexcitation. For example, fluorescent tags can be used as labels forlabelling the target samples. Upon excitation, the fluorescent tags arecaused to emit fluorescent light having a characteristic emissionspectrum. The detection of this characteristic emission spectrum at aparticular spot indicates that the labelled target molecule has bound tothe particular type of binding sites present at the respective spot.

A sensor for detecting labelled target samples is described in thearticle “Zeptosens' protein microarrays: A novel high performancemicroarray platform for low abundance protein analysis”, Proteomics2002, 2, S. 383-393, Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany. Thesensor described there comprises a planar waveguide arranged on asubstrate, and a grating for coupling coherent light of a predeterminedwavelength into the planar waveguide. A further grating is arranged atthat end of the planar waveguide remote from the grating for couplingthe light into the waveguide. Coherent light that has propagated throughthe planar waveguide is coupled out of the waveguide by the furthergrating. The outcoupled light is used for adjustment of the coupling ofcoherent light of predetermined wavelength into the planar waveguide.The coherent light propagates through the planar waveguide under totalreflection with an evanescent field of the coherent light propagatingalong the outer surface of the planar waveguide. The depth ofpenetration of the evanescent field into the medium of lower refractiveindex at the outer surface of the planar waveguide is in the order ofmagnitude of a fraction of the wavelength of the coherent lightpropagating through the planar waveguide. The evanescent field excitesthe fluorescent tags of the labelled target samples bound to the bindingsites arranged on the surface of the planar waveguide. Due to the verysmall penetration of the evanescent field into the optically thinnermedium at the outer surface of the planar waveguide, only the labelledsamples bound to the binding sites immobilized on the outer surface ofthe planar waveguide are excited. The fluorescent light emitted by thesetags is then detected with the aid of a CCD camera.

SUMMARY

While it is principally possible to detect the binding affinities usingfluorescent labels, this technique is disadvantageous in that thedetected signal is produced by the labels rather than by the bindingpartners themselves. In addition, labelling the target samples requiresadditional working steps. Moreover, labelled target samples arecomparatively expensive. Another disadvantage is the falsification ofthe results caused by photobleaching or quenching effects.

It is an object of the present invention to provide a device for use inthe detection of binding affinities of a target sample as well as asystem and a method capable of detecting such binding affinities whichovercome or at least greatly reduce the disadvantages of the prior artsensor described above.

In accordance with the invention, this object is achieved by a devicefor use in the detection of binding affinities. The device comprises aplanar waveguide arranged on a substrate, and further comprises anoptical coupler for coupling coherent light of a predeterminedwavelength into the planar waveguide such that the coherent lightpropagates through the planar waveguide with an evanescent field of thecoherent light propagating along an outer surface of the planarwaveguide. The outer surface of the planar waveguide comprises bindingsites thereon capable of binding target samples to the binding sitessuch that light of the evanescent field is scattered by target samplesbound to the binding sites. The binding sites are arranged along aplurality of predetermined lines, the predetermined lines being arrangedsuch that the light scattered by the target samples bound to the bindingsites interferes at a predetermined detection location with a differencein optical path length which is an integer multiple of the predeterminedwavelength of the light.

The detection of binding affinities according to the invention isneither limited to specific types of target samples nor to any type ofbinding sites, but rather the binding characteristics of molecules,proteins, DNA etc. can be analysed with respect to any type of bindingsites on the planar waveguide. The detection of binding affinities canbe achieved in a label-free manner. Alternatively, scattering enhancers(e.g. scattering labels) which strongly scatter the light can be used toincrease the detection sensitivity. Such scattering enhancers can be ananoparticle (alone or with a binder) or in another example a colloidalparticle. The binding characteristic to be analysed can be of statictype (for example, it can be analysed whether a target sample has or hasnot bound to the binding sites) or of dynamic type (for example, thedynamics of the binding process over time can be analysed). Bindingsites are locations on the outer surface of the planar waveguide towhich a target sample may bind. For example, binding sites may comprisecapture molecules which are immobilized on the outer surface of theplanar waveguide, or may simply comprise activated locations on theouter surface of the planar waveguide which are capable of bindingtarget samples to the activated locations, or may be embodied in anyother manner suitable to bind target samples at the desired locations onthe outer surface of the planar waveguide. The plurality ofpredetermined lines may comprise individual separate lines or maycomprise a line pattern in which the individual lines are connected toform a single line, for example a meandering single line pattern. Thedistance between adjacent predetermined lines along which the bindingsites are arranged is chosen with respect to the predeterminedwavelength of the light. Preferred distances between adjacentpredetermined lines are of the order of more than 100 nm. A range ofabout 100 nm to about 1000 nm for the distance between adjacentpredetermined lines is preferred for the use of visible light in theplanar waveguide so that the scattered light can be detected by standardoptical means. In addition, it is preferred that the planar opticalwaveguide has a high refractive index relative to the medium on theouter surface of the planar waveguide, so that the penetration depth ofthe evanescent field is only small and the fraction of coherent lightpropagating in the evanescent field is high. For example, the refractiveindex of the planar waveguide may be in the range of 1.6 to 2.5, whereasthe refractive index of the medium at the surface of the planarwaveguide is typically in the range of 1 to 1.5. By way of example, thebinding sites may comprise capture molecules which are immobilised onthe outer surface of the planar waveguide. The immobilized capturemolecules together with the target samples bound thereto form aplurality of scattering centres scattering the coherent light of theevanescent field. The coherent light propagating along the planarwaveguide has a predetermined wavelength and is preferably monochromatic(ideally at a single wavelength). Since the light of the evanescentfield propagating along the surface of the planar waveguide is coherentas is the light propagating within the planar waveguide, the coherentlight of the evanescent field is scattered coherently by the scatteringcentres formed by the target molecules bound to the capture molecules(or more generally, by the target sample bound to the binding sites)which are arranged on the different predetermined lines. The scatteredlight at any location can be determined by adding the contributions fromeach of the individual scattering centres. A maximum of the scatteredlight is located at the predetermined detection location because thepredetermined lines are arranged such that at the predetermineddetection location, the optical path length of the light scattered bythe different scattering centres differs by an integer multiple of thewavelength of the light. For a maximum signal at the detection location,the optical path length of the light from the optical coupler to thepredetermined lines and from there to the predetermined detectionlocation is also a multiple integer of the predetermined wavelength.Thus the light scattered by the target samples bound to the bindingsites interferes at a predetermined detection location. The requirementof constructive interference is met by any scattered light which adds tothe detectable signal in the detection location. The predetermineddetection location is not limited to a particular shape, for example itmay have the shape of a point or a strip. The arrangement of the bindingsites “along the predetermined lines” represents the optimum case inwhich all binding sites are exactly arranged on the predetermined lines.Such optimal arrangement of the binding sites results in a maximumsignal at the detection location. It is obvious to the person skilled inthe art that in practice the arrangement of the binding sites candeviate to some extent from such optimum arrangement. For example, thedeviation may be caused by the method for arranging the binding sites onthe outer surface of the planar waveguide, as will be explained in moredetail below.

In accordance with one aspect of the device according to the invention,the distance between adjacent predetermined lines decreases in thedirection of propagation of the light of the evanescent field. Ingeneral, the angles under which the scattered light of the evanescentfield interferes at the predetermined detection location are differentfor the various scattering centres (target samples bound to the bindingsites), which are arranged along the predetermined lines. Since at thepredetermined detection location the scattered light is to interfere toa maximum, the difference in optical path length of the light scatteredfrom the various scattering centres must be a multiple integer of thewavelength of the light. The decrease in distance between the adjacentpredetermined lines takes account of that fact and causes the light tointerfere to a maximum at the predetermined detection location, whichdoes not need to have the shape of a point or a small spot but may alsohave the shape of a strip or any other desired shape.

According to a further aspect of the device according to the invention,the plurality of predetermined lines on which the binding sites arearranged comprises curved lines. The curvature of the lines is such thatlight of the evanescent field scattered by the target samples bound tothe binding sites arranged along these predetermined lines interferes toa maximum at the predetermined detection location. The detectionlocation preferably has the shape of a point. Each of the individualpredetermined lines may have a curvature which is different from thecurvature of the other predetermined lines. In practice, the detectionlocation is not a point but may be a small spot or a strip having alength which is smaller than the length of the predetermined lines alongwhich the binding sites are arranged. The curvature of each individualcurved predetermined line is chosen such that the optical path length ofthe light propagating from the optical coupler to the individualpredetermined line and from there to the predetermined detectionlocation is a multiple integer of the predetermined wavelength of thepropagating light for the entire curved line. This is advantageous inthat also the light scattered by scattering centres located on the outersections of the predetermined lines contributes to the signal in thespatially reduced area of the point-shaped (or spot or strip-shaped)detection location.

In accordance with still a further aspect of the device according to theinvention, the plurality of predetermined lines are arranged on theouter surface of the planar waveguide in a manner such that theirlocations in x_(j),y_(j)-coordinates are geometrically defined by theequation

$x_{j} = \frac{{\lambda\;{N\left( {A_{0} + j} \right)}} - \sqrt{{{n_{s}^{2}\left( {N^{2} - n_{s}^{2}} \right)}\left( {y_{j}^{2} + f^{2}} \right)} + {\left( {n_{s}\lambda} \right)^{2}\left( {A_{0} + j} \right)^{2}}}}{N^{2} - n_{s}^{2}}$wherein

-   λ is the vacuum wavelength of the propagating light,-   N is the effective refractive index of the guided mode in the planar    waveguide; N depends on the thickness and the refractive index of    the planar waveguide, the refractive index of the substrate, the    refractive index of a medium on the outer surface of the planar    waveguide and the polarization of the guided mode,-   n_(s) is the refractive index of the substrate,-   f is the thickness of the substrate,-   A₀ is an integer which is chosen to be close to the product of the    refractive index n_(s) and the thickness f of the substrate divided    by the wavelength λ, and-   j is a running integer that indicates the index of the respective    line.    The chosen integer A₀ assigns negative x-values at the centre of the    lines with negative j values and positive x-values at the centre of    lines with positive j values. Or to say it in other words, the    integer A₀ defines the origin of the x,y-coordinates frame that is    used for the location of the lines at the outer surface of the    planar waveguide; the chosen A₀ value puts the detection location at    x=0, y=0, z=−f.

As already outlined above, for an improved signal at the predetermineddetection location it is preferred that the plurality of predeterminedlines are arranged in a manner such that the scattering centres arrangedalong these predetermined lines are located on a curved grid-likestructure with a decreasing distance between adjacent predeterminedlines. Such an arrangement fulfils the condition that the difference inoptical path length for the light propagating from the optical couplerto the individual predetermined lines and scattered by the scatteringcentres to the predetermined detection location is a multiple integer ofthe predetermined wavelength of the light propagating in the waveguide.Also, the optical path length of the light propagating from the opticalcoupler to the individual predetermined lines and from there to thepredetermined detection location is a multiple integer of thepredetermined wavelength of the propagating light for the entire curvedline. Thus, it is possible to form a compact device due to the bindingsites being arranged on the surface of the planar waveguide while thedetection location may be formed at the bottom surface of the substratecarrying the planar waveguide.

Two embodiments are particularly envisaged of how the binding sites canbe arranged along the plurality of predetermined lines. According to afirst embodiment, the binding sites comprise capture molecules attachedto the surface of the planar waveguide along the predetermined linesonly. These capture molecules are capable of binding the target samplesand are immobilized on the outer surface of the planar waveguide(although, as mentioned above, the binding sites can be formed by theactivated surface of the planar waveguide itself). Immobilizing thecapture molecules on the outer surface of the planar waveguide along thepredetermined lines can generally be performed by any suitable method,for example it may be performed using photolithographic methods using alithographic mask with curved lines. It goes without saying, that thearrangement of the binding sites along the predetermined lines is to beunderstood in any embodiment of the invention in a sense that themajority of the binding sites—in the instant embodiment the capturemolecules—are located along the predetermined lines and does explicitlyinclude that some binding sites are arranged at locations differenttherefrom.

According to a second embodiment, the binding sites again comprisecapture molecules capable of binding the target samples, which is norestriction to a certain type of binding site or a certain type oftarget sample. The capture molecules are again capable of binding thetarget samples. However, the arrangement of capture molecules capable ofbinding the target molecules along the predetermined lines is performedby dispensing and immobilizing capture molecules capable of binding thetarget samples on the (entire) surface of the planar waveguide, and bysubsequently deactivating those capture molecules which are not arrangedalong the predetermined lines. The term “deactivating” in this respectrefers to any suitable method for changing the binding capability of thecapture molecules (for example by exposing the capture molecules tolight for a predetermined time) in order to achieve that they are nolonger capable of binding target samples. According to this embodimentof the invention, the capture molecules can be applied uniformly orstatistically onto the outer surface of the planar waveguide. Afterdeactivation of capture molecules which are arranged between thepredetermined lines only the capture molecules arranged along thepredetermined lines (these have not been deactivated) are capable ofbinding a target sample. Nevertheless, the deactivated capture moleculesremain immobilized on the outer surface of the planar waveguide.

This embodiment has the additional advantage that the contribution ofthe signal generated by the light scattered by target molecules bound tocapture molecules to the overall signal at the detection location isincreased. Generally, the difference between the signals of the lightscattered by the target molecules bound to the captures molecules andthe light scattered by the capture molecules without any targetmolecules bound thereto is small compared to the light scattered by thecapture molecules alone. Assuming that the scattering properties of thecapture molecules arranged along the predetermined lines (which have notbeen deactivated) and of the deactivated capture molecules arrangedbetween the predetermined lines are identical and further assuming thatthe capture molecules are homogeneously distributed over the outersurface of the planar waveguide, then ideally no signal is produced atthe detection location after the capture molecules have been immobilizedon the outer surface of the planar waveguide and after the capturemolecules arranged between the predetermined lines have beendeactivated. In practice, however, deactivation of the capture moleculesslightly changes the scattering properties of the capture molecules, sothat it may not be ideal to deactivate all of the capture moleculeswhich are arranged between the predetermined lines. Instead, only thevast majority of the capture molecules arranged between thepredetermined lines may be deactivated. Deactivation of the capturemolecules is performed to an extent such that the overall signal at thedetection location produced by those capture molecules arranged alongthe predetermined lines and by those deactivated and the fewnon-deactivated capture molecules arranged between the predeterminedlines is at a minimum, and is preferably zero. Assuming that the signalso obtained at the detection location can be reduced to zero this means,that after adding the target samples the signal produced at thedetection location only results from target samples bound to the capturemolecules. In case no target samples are bound to the capture molecules,the signal at the detection location remains zero. This increases thesensitivity of the detector for the signal generated by the lightscattered by the target molecules bound to the capture molecules at thedetection location.

In accordance with a further aspect of the device according to theinvention, the planar waveguide has a refractive index n_(w) which issubstantially higher than the refractive index n_(s) of the substrateand which is also substantially higher than the refractive index n_(med)of the medium on the outer surface of the planar waveguide, such thatfor a predetermined wavelength of the light the evanescent field has apenetration depth in the range of 40 nm to 200 nm. The term“substantially higher” shall be understood as designating a differencein refractive index allowing a coupling in of the light into the planarwaveguide where it propagates under total reflection. The lightpropagating along the planar waveguide has an evanescent field whichpropagates along the outer surface of the planar waveguide. Theevanescent field has a penetration depth which depends on the indexn_(med), the effective refractive index N of the guided mode, as well ason the wavelength of the propagating light, so that the penetrationdepth can be adapted such that the light of the evanescent field iscoherently scattered by the target samples bound to the binding siteslocated on (or in proximity) to the predetermined lines on the outersurface. The approximate values of the penetration depth mentioned aboveare to be understood to explicitly include the exact boundary valuesthereof.

In accordance with a further aspect of the device according to theinvention, the device comprises a further optical coupler for couplingout the light that has propagated through the planar waveguide. Both,the optical coupler for coupling the light into the planar waveguide aswell as the further optical coupler for coupling out the light that haspropagated through the planar waveguide may comprise an optical gratingfor coherently coupling light into and out of the planar waveguide. Theoptical coupler and the further optical coupler comprise an opticalgrating for coherently coupling light into and out of the planarwaveguide under a respective predetermined in-coupling angle orout-coupling angle. The in-coupling angle or out-coupling angle isdetermined by the wavelength of the light as well as by thecharacteristic of the optical coupler. However, within the scope of theinvention the light can also be coupled into and out of the planarwaveguide by any other means suitable for coupling light into and out ofa planar waveguide of a thickness in the range of some nanometers tosome hundred nanometers. Only by way of example, an alternative opticalcoupler may be an optical prism.

In accordance with a further aspect of the device of the invention, theplanar waveguide has a first end section and a second end section whichare arranged at opposite ends of the planar waveguide with respect tothe direction of propagation of the light through the planar waveguide.The first end section and the second end section comprise a materialwhich is absorptive at the wavelength of the light propagating throughthe planar waveguide. The absorptive material minimizes reflections ofthe light propagating along the planar waveguide towards the respectiveend section and back into the planar waveguide. This improves thedetected signal as light which may have been reflected from the ends ofthe planar waveguide is eliminated or at least greatly minimized.

In accordance with a further aspect of the device according to theinvention, a plurality of measurement zones are arranged on the outersurface of the planar waveguide. In each measurement zone the bindingsites are arranged along the plurality of predetermined lines. Forhigh-throughput screening, the simultaneous detection of bindingaffinities of a sample can be achieved for different types of bindingsites and target samples by arranging the respective target samplesbound to the binding sites in separate measurement zones. Eachmeasurement zone has a corresponding individual detection location toallow a separate detection of the scattered light of the evanescentfield.

In accordance with a further aspect of the device according to theinvention, the plurality of measurement zones comprises measurementzones of different sizes. All sizes of the measurement zones are known.At the respective detection location, the light scattered incorresponding measurement zones of different size in which the same typeof target samples is bound to the same type of binding sites can becompared. The intensity of the scattered light at the detection locationhas a quadratic correlation to the number of scattering centers in therespective measurement zone on the planar surface of the waveguide.Thus, for a uniform distribution and areal density of scattering centersin the measurement zones of different sizes, the intensities of thescattered light at the respective detection locations of correspondingmeasurement zones of different sizes have a quadratic correlation to thesize of the respective measurement zones. Therefore, the intensities ofthe scattered light at detection locations of measurement zones ofdifferent sizes can be used to verify that the measured intensities areindeed representative of light scattered by the scattering centersarranged on the predetermined lines.

According to an aspect of the invention, each measurement zone has anarea larger than 25 μm², wherein the plurality of predetermined lineshas a distance between adjacent predetermined lines less than 1.5 μm, inparticular less than 1 μm. This allows to achieve highly integrateddevices with high numbers of measurement zones, i.e. 1000, 10000,100000, . . . , up to 4×10⁶ measurement zones per square centimetre.

Advantageously, the binding sites are arranged along at least twopluralities of predetermined lines in a single measurement zone. Each ofthe two pluralities of predetermined lines is arranged such that thelight scattered by the target samples bound to the binding sitesarranged along the respective plurality of predetermined linesinterferes with a difference in optical path length which is an integermultiple of the predetermined wavelength of the light at an individualdetection location for each of the plurality of predetermined lines. Theindividual detection locations are spatially separated from each other.More than one plurality of predetermined lines in the measurement zonewhich are arranged so as to provide spatially separated detectionlocations allow carrying out additional methods for the detection ofbinding events (e.g. the detection of cooperative bindings or thedetection of reaction cascades).

In accordance with another aspect of the device according to theinvention, the device comprises a diaphragm having an aperture which isarranged such that light at the detection location is allowed to passthrough the aperture while light at a location different from thedetection location is blocked by the diaphragm. A mechanical diaphragmas well as an electronic diaphragm can be adapted to blind out all lightother than that being scattered to the detection location.Advantageously, the diaphragm can be formed on the outer surface of thesubstrate on that side remote from the planar waveguide. For example, anon-transparent material, e.g. a chromium layer, can be applied to thesurface of the substrate remote to the waveguide. The non-transparentchromium layer has a transparent aperture at the detection locationthrough which the light scattered to the detection location can passwhile the rest of the light not scattered to the aperture is blocked.

In accordance with a still further aspect of the device according to theinvention, the diaphragm further comprises at least one further aperturewhich is arranged adjacent to the aperture when viewed in the directionof propagation of the light through the planar waveguide. The furtheraperture is located adjacent the aperture such that incoherent lightscattered to the further aperture may pass through the further aperture.Advantageously, the detected incoherent background light can becorrected by the use of a diaphragm having a further aperture. Thefurther aperture does not itself detect the incoherent background lightat the detection location but allows determination of the amount of theincoherent light at the detection location from a measurement of theincoherent light at a location different from the detection location.The so determined amount of incoherent light at the detection locationcannot be separated from the light at the detection location, but can besubtracted from the entire signal at the detection location once theentire signal at the detection location has been measured by a detector.For an improved correction a first further aperture is located withrespect to the direction of the propagating light in front of thedetection location and a second further aperture is located behind thedetection location. Such a configuration allows detecting an averagevalue for the incoherent light at the detection location for correctingthe signal at the detection location.

Another aspect of the invention relates to a system for the detection ofbinding affinities comprising a device for the detection of bindingaffinities according to the invention. The system further comprises alight source for emitting coherent light of a predetermined wavelength,the light source and the device being arranged relative to one anothersuch that the coherent light is coupled into the planar waveguide viathe optical coupler. Alternatively, the system further comprises opticalmeans for scanning and/or adjustment of the angle of light impinging onthe optical coupler since the exact coupling angle of the opticalcoupler can vary from device to device. Alternatively, the wavelength ofthe light emitted by the light source in the system can be tuned whichmay be advantageous in case the angle of the light impinging on theoptical coupler is fixed for constructional reasons.

According to still another aspect of the system according to theinvention, the system further comprises an optical imaging unit, theoptical imaging unit being focused such as to produce an image of thedetection location of the device. The optical imaging unit is capable ofproviding an image of the predetermined detection location in which thelight scattered by the target samples bound to the binding sitesinterferes with a difference in optical path length which is an integermultiple of the predetermined wavelength of the light. The opticalimaging unit can be used for imaging the light present at the detectionlocation to an observation location. The optical imaging unit can beadapted for imaging both the light from the detection location as wellas the light from the further aperture or further apertures, since thislight may be used for subtraction of the incoherent background lightfrom the entire light present at the detection location. Alternativelyor in addition, the optical imaging unit can be used to select only thelight at the detection location by focusing the optical imaging unit tothe detection location. A diaphragm is then no longer needed.

Another aspect of the invention relates to a method for detection ofbinding affinities. The method comprises the steps of:

providing a device comprising a planar waveguide arranged on a substrateand an optical coupler,

coupling coherent light of a predetermined wavelength into the planarwaveguide such that the coherent light propagates along the planarwaveguide with an evanescent field of the coherent light propagatingalong an outer surface of the planar waveguide,

attaching target samples to binding sites arranged along a plurality ofpredetermined lines on the outer surface of the planar waveguide,

detecting, at a predetermined detection location, light of theevanescent field scattered by target samples bound to binding sitesarranged along the predetermined lines, the light scattered by thetarget samples bound to the binding sites having, at the predetermineddetection location, a difference in optical path length which is aninteger multiple of the predetermined wavelength of the light.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantageous aspects of the invention become apparent from thefollowing description of embodiments of the invention with reference tothe accompanying schematic drawings in which:

FIG. 1 shows a perspective view of an embodiment of the device accordingto the invention;

FIG. 2 shows a sectional view of the device of FIG. 1;

FIG. 3 shows an illustration of different optical paths for the light ofthe evanescent field propagating along the outer surface and beingscattered to the detection location;

FIG. 4 shows a measurement zone of the device according to the inventioncomprising an arrangement of a plurality of predetermined lines, withbinding sites being immobilized along the predetermined lines;

FIG. 5 shows the measurement of FIG. 4, with some target samples beingbound to the binding sites;

FIG. 6 shows a measurement zone of a device according to the inventioncomprising an arrangement of a plurality of predetermined lines, withbinding sites being immobilized along the predetermined lines andbetween the predetermined lines;

FIG. 7 shows the measurement zone of FIG. 6, with those binding sitesarranged between the predetermined lines being deactivated;

FIG. 8 shows the measurement zone of FIG. 7 with the target samplesbeing added;

FIG. 9 shows the measurement zone of FIG. 8 with the target samplesbeing bound to the binding sites immobilized along the predeterminedlines;

FIG. 10 shows an illustration of the construction of a blank section inwhich the predetermined lines of a measurement zone are to beeliminated;

FIG. 11 shows the measurement zone of FIG. 10, with a blank section inwhich the predetermined lines are eliminated;

FIG. 12 shows a top view of a further embodiment of the device accordingto the invention comprising a plurality of measurement zones;

FIG. 13 shows a bottom view of the embodiment of the device of FIG. 10,with an aperture being provided at the detection location and twofurther apertures being provided at locations before and behind thedetection location for each measurement zone;

FIGS. 14-17 show a portion of a measurement zone of a device accordingto the invention in different phases of a process of binding targetsamples;

FIG. 18 shows a sectional view of a further embodiment of the device inwhich the device comprises an additional carrier substrate;

FIG. 19 shows an illustration of different optical paths for the lightof the evanescent field scattered at two different pluralities ofpredetermined lines arranged in a single measurement zone;

FIG. 20 shows a top view of the device of FIG. 18 having twelvemeasurement zones arranged thereon, with three pluralities ofpredetermined lines being arranged in each measurement zone; and

FIG. 21 shows a bottom view of the device of FIG. 18 with apertures in anon-transparent layer formed on top of the additional carrier substrate.

DETAILED DESCRIPTION

FIG. 1 shows a perspective view of an embodiment of the device accordingto the invention for the detection of binding affinities of a sample.The device comprises a substrate 3 of a transparent material which inthe embodiment shown has the shape of a rectangular cube, without beinglimited to this shape. A planar waveguide 2 (see also FIG. 2) isarranged on the upper side of the substrate 3, into which a coherentlight 1 is coupled such that the coherent light propagates through theplanar waveguide 2 under total reflection. Since the planar waveguide 2has a thickness in the range of some nanometers to some hundrednanometers only, it is not illustrated as a separate layer in FIG. 1,but is shown exaggerated in FIG. 2. As illustrated by the parallelarrows in FIG. 1, coherent light 1 of a predetermined wavelength iscoupled through the substrate 3 into the planar waveguide 2 with the aidof a grating 4 acting as an optical coupler. The coherent light coupledinto the planar waveguide 2 propagates along the planar waveguide 2 withan evanescent field 6 (represented by an arrow) penetrating into themedium above the upper surface of the planar waveguide 2 (see again FIG.2). The evanescent field 6 propagates along the outer surface 5 of theplanar waveguide 2. A measurement zone 10 arranged on the outer surfaceof the planar waveguide 2 comprises a plurality of predetermined lines 9(each of the shown lines represent a multiplicity of lines, inparticular fifty lines in the present example of such a device; and onlyone such measurement zone being shown in FIG. 1 for the sake ofclarity). Binding sites (not shown in FIG. 1) to which target samplescan bind are arranged along these predetermined lines 9. Coherent lightof the evanescent field 6 is scattered by the target samples bound tobinding sites within the measurement zone 10. Some of the lightscattered by the target samples bound to binding sites is directed to adetection location where a diaphragm 11 comprising an aperture 21 isarranged. The diaphragm 11 is made from a non-transparent material andmay for example be a chromium layer which is applied onto the lowersurface of the substrate 3.

FIG. 2 shows a sectional view of the device of FIG. 1 with the thicknessof the planar waveguide being shown exaggerated for the purpose ofexplaining the general working principle. As can be seen, the lightcoupled into the planar waveguide 2 with the aid of optical grating 4propagates through the planar waveguide 2 under total reflection untilreaching a further grating 13 arranged at the opposite end of the planarwaveguide 2. This further grating serves as a further optical coupler tocouple the light out of the planar waveguide. To avoid reflections andfor minimizing incoherent background light, a first end section 14 and asecond end section 15 of the planar waveguide 2 comprise an absorptivematerial. Corresponding to the light propagating in the planarwaveguide, the evanescent field 6 propagates along the outer surface 5of the planar waveguide 2.

The refractive index n_(w) of the planar waveguide 2 is substantiallyhigher than the refractive index n_(s) of the substrate 3 and alsosubstantially higher than the refractive index n_(med) of the medium onthe outer surface 5 of the planar waveguide 2. The refractive indexn_(med) of the medium on the outer surface 5 may vary depending on thetype of sample applied thereto. For example, the refractive indexn_(med) for the medium on the outer surface 5 can be of the order of therefractive index of water in case the target sample is present in anaqueous solution applied to the outer surface 5 of the planar waveguide2, or may be of the order of the refractive index of air in case of drytarget samples, or may be in the order of the refractive index of ahydrogel layer 16 in case the binding sites to which target samples 8can bind are contained in a hydrogel layer 16 on the outer surface 5.The penetration depth of the evanescent field 6 into the medium on theouter surface 5 of the planar waveguide 2 (distance between the outersurface 5 of the planar waveguide 2 and the 1/e² intensity descent ofthe evanescent field 6) depends on the index n_(med) of the medium onthe outer surface 5 of the planar waveguide 2, the effective refractiveindex N of the guided mode and on the wavelength λ of the light.

The light in the evanescent field 6 propagating along the outer surface5 of the planar waveguide 2 is scattered by target samples 8 bound tobinding sites, and these binding sites may comprise capture molecules 7which are capable of binding the target samples 8 and which are arrangedin the measurement zone 10 along the predetermined lines 9 (FIG. 1). InFIG. 2 it is indicated by arrows of decreasing length, that the distancebetween adjacent predetermined lines along which the capture molecules 7are arranged decreases when viewed in the direction of propagation ofthe light. As can further be seen, in the embodiment shown in FIG. 2 thetarget samples 8 have been applied to the measurement zone by dispensinga droplet containing the target samples 8. Some of the light scatteredby the target samples 8 bound to the capture molecules 7 is directed tothe detection location where the aperture 21 of diaphragm 11 isarranged. As an option, the light at the detection location can beimaged on a photo-detector 20 by an optical imaging unit 19. The opticalimaging unit 19 and the photo-detector 20 are shown surrounded by a boxdrawn in dashed lines, since they can be provided alternatively or incombination, and can in particular be provided in combination in oneunit.

While it is already evident from FIG. 2 that the lengths of the opticalpaths of the light of the evanescent field 6 which is scattered bytarget samples 8 bound to different capture molecules 7 to the detectionlocation is different, this becomes even more clear when glancing atFIG. 3 in which a number of such different optical paths are explicitlyshown. Some of the coherent light of the evanescent field 6 is scatteredby the target samples 8 bound to different capture molecules 7 in amanner such as to interfere at the detection location which is thelocation of the aperture 21 of diaphragm 11. For a predetermineddetection location, the arrangement and geometry of the predeterminedlines 9 as well as the thickness of the substrate 3 are selected suchthat at the detection location the difference in optical path length isan integer multiple of the predetermined wavelength of the coherentlight. Thus, the interference of the light at the detection location isthe coherent additive superposition of the light scattered to thedetection location by the target molecules 8 bound to the differentcapture molecules 7.

For the embodiment shown in FIG. 3 the plurality of predetermined curvedlines 9 are arranged on the outer surface 5 of the planar waveguide in amanner such that their locations in the plane of the outer surface 5 ofthe planar waveguide are geometrically expressed inx_(j),y_(j)-coordinates by the equation

$x_{j} = \frac{{\lambda\;{N\left( {A_{0} + j} \right)}} - \sqrt{{{n_{s}^{2}\left( {N^{2} - n_{s}^{2}} \right)}\left( {y_{j}^{2} + f^{2}} \right)} + {\left( {n_{s}\lambda} \right)^{2}\left( {A_{0} + j} \right)^{2}}}}{N^{2} - n_{s}^{2}}$wherein

-   λ is the vacuum wavelength of the propagating light,-   N is the effective refractive index of the guided mode in the planar    waveguide; N depends on the thickness and the refractive index of    the planar waveguide, the refractive index of the substrate, the    refractive index of a medium on the outer surface of the planar    waveguide and the polarization of the guided mode,-   n_(s) is the refractive index of the substrate,-   f is the thickness of the substrate,-   A₀ is an integer which is chosen to be close to the product of the    refractive index n_(s) and the thickness f of the substrate divided    by the wavelength λ, and-   j is a running integer that indicates the index of the respective    line.

FIG. 4 shows a measurement zone 10 in an enlarged view comprising thepredetermined lines 9 and binding sites represented by capture molecules7 which are immobilized on the outer surface of the planar waveguide 5(see FIG. 1) along the predetermined lines 9. Immobilizing the capturemolecules 7 along the predetermined lines only can be performed with theaid of lithographic techniques, as this has been discussed above. InFIG. 5 target samples 8 are bound to some of the capture molecules 7.Since the capture molecules 7 are arranged along the plurality ofpredetermined lines 9, target samples 8 bound to the capture molecules 7are arranged along the plurality of predetermined lines 9, too. At thedetection location this results in the coherent additive superpositionof the light scattered by the scattering centres formed by the targetsamples 8 bound to the capture molecules 7, as this has been explainedabove.

FIG. 6, FIG. 7, FIG. 8 and FIG. 9 show again a measurement zone 10 in anenlarged view. However, the manner how the capture molecules 7 capableof binding the target samples 8 have been immobilized along thepredetermined lines 9 is different.

As can be seen in FIG. 6, in a first step the capture molecules 7 areimmobilised over the (entire) outer surface of the planar waveguide inthe measurement zone 10, so that there is no arrangement of the capturemolecules along the plurality of predetermined lines 9. Thus, the lightof the evanescent field scattered by the capture molecules 7 does notinterfere at the detection location in the manner described above.

As can be seen in FIG. 7, the capture molecules arranged between thepredetermined lines 9 have been deactivated so that no target samplescan bind to these deactivated capture molecules 12 anymore. Accordingly,the only capture molecules 7 capable of binding target samples arearranged along the plurality of predetermined lines 9. The accuracy ofthe immobilization of the capture molecules 7 along the predeterminedlines 9 depends on the method of attaching, immobilizing or deactivatingthe capture molecules 7. As a result, the location of the immobilizedcapture molecules 7 capable of binding the target samples 8 may not beexactly “on” the predetermined lines 9 but may to some extent deviatefrom the exact location “on” the predetermined lines 9. In practice, thedeviation from the exact location “on” the predetermined lines may bewithin a range which is smaller than a quarter of the distance ofadjacent predetermined lines 9. This results in a still constructiveinterference of the light scattered to the detection location.

As has been explained in the introductory part, deactivation of thecapture molecules 12 arranged between the predetermined lines 9 isperformed such that after deactivation the overall signal at thedetection location (no target samples 8 have been added yet) produced bythe deactivated capture molecules 12 and the capture molecules 7 capableof binding the target samples 8 is set or adjusted to a tuned minimumsignal at the detection location, ideally to zero.

The next step is adding the target samples 8 to the measurement zone 10on the outer surface of the planar waveguide, as this is shown in FIG.8. Since only the capture molecules 7 arranged along the predeterminedlines 9 are capable of binding the target samples 8, the target samples8 are bound to those capture molecules 7 along the predetermined lines9, as this is shown in FIG. 9. Due to the tuned signal at the detectionlocation caused by the deactivated capture molecules 12 and the capturemolecules 7 having been set or adjusted to a minimum before (see above),the signal at the detection location is then mainly (or entirely, if thesignal produced by deactivated capture molecules and the capturemolecules has been reduced to zero before) caused by the light scatteredby the target samples 8 bound to the capture molecules 7 arranged alongthe predetermined lines.

FIG. 10 shows a portion of measurement zone 10 as described above forillustrating the construction of a blank section in which thepredetermined lines 9 are to be eliminated to avoid second order Braggreflections in the planar waveguide. Bragg reflections are to be avoidedsince they result in a reduction in intensity of the light propagatingalong the planar waveguide. This is particularly disadvantageous in casea plurality of measurement zones 10 is arranged one after the other onthe outer surface of the planar waveguide in the direction of thepropagating light. Thus, a decrease of the intensity of the propagatinglight scattered in the subsequent measurement zones is not only due thedescribed scattering processes in the various measurement zones butadditionally decreases due to Bragg reflections in the planar waveguide.Since in each of the measurement zones the predetermined lines 9 in acircular section of the measurement zone have a distance betweenadjacent lines which fulfils the condition for second order Braggreflection, the Bragg reflection of second order in the planar waveguidedefines a further location 22 at which the Bragg reflected lightconstructively interferes. In the shown example, the intersection pointsof the shown arc of circle 21 with the predetermined lines 9 indicatesthose locations of the predetermined lines 9 for which the Braggcondition is exactly fulfilled, so that light is reflected back andinterferes constructively at the further location 22. This reflectedlight is not available for scattering in subsequently arrangedmeasurement zones 10.

FIG. 11 shows a measurement zone 10 comprising a region 23 in proximityto the arc of circle 21 where the predetermined lines 9 are eliminatedto avoid such second order Bragg reflections.

FIG. 12 and FIG. 13 show top and bottom views of a further embodiment ofthe device according to the invention. This embodiment of the devicecomprises a plurality of measurement zones 10 of a first size andmeasurement zones 17 of a different size. Each measurement zone 10comprises a region 23 in which the plurality of predetermined lines 9 iseliminated to avoid Bragg reflections (see above). Generally, it is alsopossible that the measurement zones do not comprise the regions 23. Anoptical grating 4 for coupling light into the planar waveguide and afurther grating 13 for coupling the light out of the planar waveguideare provided. Between the optical grating 4 and the further opticalgrating 13 a plurality of measurement zones 10 of the first size and ofmeasurement zones 17 of different size are arranged where binding sitesare arranged along the predetermined lines 9, as this has been discussedin detail above. The plurality of measurement zones 10 of the first sizeand the plurality of measurement zones 17 of different size allows thesimultaneous detection of different combinations of target samples andbinding sites, so that a plurality of combinations of target samples andbinding sites can be analysed simultaneously with respect to the bindingaffinity of specific target samples to specific binding sites.Alternatively, redundant measurements can be carried out for the samecombinations of target samples and binding sites.

From the bottom view of FIG. 13 it can be seen that an aperture 21 isprovided for each measurement zone at the detection location, where thescattered light has a difference in optical path length which is amultiple integer of the wavelength of the light propagating in thewaveguide to the scattering centre on a predetermined line and fromthere to the predetermined detection location, as this has also beendiscussed in detail above. It goes without saying that an opticalimaging unit may be provided as this has been discussed in detail withrespect to FIG. 2.

The measurement zones 17 of different size are arranged in between themeasurement zones 10. The measurement zones 17 have a known sizedifferent from the size of the measurement zone 10, all sizes beingknown. At the respective detection location, the light scattered inmeasurement zones 10 and in corresponding measurement zones 17 can becompared (for the same type of target sample bound to the same type ofbinding sites). The intensity of the scattered light at the detectionlocation has a quadratic correlation to the number of scattering centersin the measurement zone on the planar surface of the waveguide. Assuminga uniform distribution and areal density of scattering centers in themeasurement zones of different sizes, the intensities of the scatteredlight at the respective detection locations of corresponding measurementzones of different sizes has a quadratic correlation to the size of therespective measurement zones. Accordingly, the intensities of thescattered light at detection locations of measurement zones of differentsizes can be used to verify that the measured intensities are indeedrepresentative of light scattered by the scattering centers arranged onthe predetermined lines.

For an improved detection of binding affinities, two further apertures18 are formed on the substrate 3 in front of and behind each aperture 21dedicated to the respective measurement zone 10. As the coherent lightpropagating through the planar waveguide 2 might be also incoherentlyscattered along its way through the planar waveguide 2, a contributionof this incoherently scattered light is also detected at the detectionlocation through the aperture 21. The apertures 18 in front of andbehind aperture 21 at a predetermined distance allow for determine anaverage signal representative of this incoherently scattered light whichcan be used to correct the detected signal at the detection location bysubtracting the average signal of the incoherent light from the overallsignal detected at the detection location. This correction of the signalat the detection location is particularly advantageous in combinationwith the afore-mentioned reduction of the background signal caused bythe scattering at the binding sites without any target moleculesattached thereto.

FIG. 14 to FIG. 17 show a portion of a measurement zone which is formedon the outer surface 5 of a planar waveguide according to the invention.Different phases of a process of binding target samples 8 to capturemolecules 7 are shown. In this process the binding of target samples 8to capture molecules 7 is enhanced. The capture molecules 7 areimmobilised at the outer surface 5. Subsequently, target samples 8 andlinkers 24 are applied. The applied target samples 8 are allowed to bindto capture molecules 7 until an equilibrium condition is reached inwhich binding of target samples 8 to capture molecules 7 and release oftarget samples 8 from capture molecules are in equilibrium. The linkeris then activated (e.g. by light) to strengthen the bindings betweentarget samples 8 and capture molecules 7. Subsequently, the non-boundtarget samples 8 as well as the unused linkers 24 are washed away. Dueto the strengthened bindings between target samples 8 and capturemolecules 7 caused by the linkers 24, inadvertent washing away of targetsamples 8 bound to capture molecules 7 is prevented or at least greatlyreduced. Thus, the signal at the detection location can be furtherenhanced. An example for such process using photo-activated linkers isdescribed in detail in “Capture Compound Mass Spectrometry: A Technologyfor the Investigation of Small Molecule Protein Interactions”, ASSAY andDrug Development Technologies, Volume 5, Number 3, 2007.

FIG. 18 shows a sectional view of a device which is principally shown inFIG. 1 but which in accordance with a further embodiment has a layerstructure to be used for example in highly integrated systems (i.e. upto about 4×10⁶ measurement zones per cm²). In the shown example, themeasurement zone 10 has an area of a size of about 25 μm². This sizeallows for arranging a multiplicity of measurement zones 10 on the outersurface 5 of the planar waveguide 2 in order to carry out a multiplicityof measurements using a single device. A measurement zone 10 of reducedsize is achieved for example by virtually “cutting out” said area ofreduced size of 25 μm² from a larger measurement zone. However, keepingthe distance between the predetermined lines in such reduced sizemeasurement zone 10 unchanged would result in that the cone formed bythe light scattered at the target samples bound to the binding sites inthe reduced size measurement zone 10 would have an aperture angle whichis substantially smaller than that of the larger size measurement zone.The smaller aperture angle of the cone of light would result in that thesame optical detection unit (comparable to FIG. 2) which has been usedfor measuring the larger measurement zone and which has a given apertureangle will measure not only light at the detection location but alsosome incoherent background light. This worsens the signal-to-noise ratio(S/N-ratio). In order to prevent this worsening of the S/N ratio, thedistance between the measurement zone 10 and the detection location mustbe reduced ideally such that the aperture angle of the cone formed bythe light scattered by the target samples bound to the binding sites ofreduced size measurement zone 10 and interfering at the detectionlocation is identical with the aperture angle of the optical detectionunit. For reducing the distance between the reduced size measurementzone 10 and the detection location, the arrangement of the plurality ofpredetermined lines in the reduced size measurement zone 10 must bedetermined according to the formula described above with reference toFIG. 3 such that the light scattered by the target samples bound to thebinding sites interferes at the new detection location. Since thedistance between the reduced size measurement zone 10 and the newdetection location is only in the range of ten micrometers (μm) to somehundred micrometers (μm) the thickness of the substrate 3 may becomeimpractically thin. In particular under laboratory conditions it may bedisadvantageous to handle devices comprising substrates 3 having athickness in the range of ten to some hundred micrometers (μm). In orderto improve the handling of such device, the device according to thisembodiment has the following layer structure (from the lower side to theupper side): an additional carrier substrate 24, a layer 111 ofnon-transparent material, the substrate 3 and the planar waveguide 2.The additional carrier substrate 24 is made of a transparent material(e.g. glass, plastic) and has a thickness rendering the device suitableto handle (e.g. up to 3 mm). The layer 111 of non-transparent materialis formed on top of the additional carrier substrate 24. The layer 111of non-transparent material is for example a black chromium layer inwhich the apertures 21, 18 are lithographically formed. The substrate 3is of a transparent material and has a thickness which corresponds tothe distance between reduced size measurement zone 10 and detectionlocation. The planar waveguide 2 and the measurement zones 10 are inprinciple similar as described further above. Each measurement zone 10may comprise more than one plurality of predetermined lines, as will bediscussed in connection with FIG. 19 below.

The illustration of optical paths in FIG. 19 is similar to FIG. 3.However, two different pluralities of predetermined lines 9, 91 arearranged in a single measurement zone, and in each such zone the lightis scattered to different spatially separated detection locations (foci)by the target samples bound to the different pluralities ofpredetermined lines 9, 91. The light of the evanescent field 6propagating along the outer surface 5 is scattered at the target samplesbound to the binding sites along the first plurality of predeterminedlines 9 such as to interfere at the right hand side focus (bold lines)and at the target samples bound to the binding sites along the secondplurality of predetermined lines 91 such as to interfere at the lefthand side focus (dashed lines). This principle applies for eachplurality of predetermined lines 9, 91 in relation to the respectivedetection location, so that additional pluralities of predeterminedlines can be arranged within such measurement zone (for example three asshown in FIG. 20). A target sample which is capable of binding tobinding sites arranged at both predetermined lines 9, 91 (FIG. 19) canform cooperative bindings via multiple bond interaction at theintersection of lines 9, 91. Such a multiple bond interaction is of ahigh strength. Both bindings can be formed simultaneously orsequentially within short periods of time (instantaneously). Suchmultiple bond interactions are optically detected at two separatedetection locations which provide correlated signals at both detectionlocations.

FIG. 20 shows a top view of the device of FIG. 18 with twelvemeasurement zones 10 arranged at the outer surface of the planarwaveguide. In each measurement zone 10, three pluralities ofpredetermined lines are provided, and the target samples bound to thebinding sites along these three pluralities of predetermined linesscatter the light coupled into the planar waveguide via the opticalcoupler 4 to three spatially separated individual detection locations.The arrangement of three pluralities is of advantage as process cascadesare detectable. Such a process cascade exists for example, when a targetsample is split up in separate products at the first type of capturemolecule arranged so as to provide a signal at the first detectionlocation. A first product of this reaction does then bind to the secondtype of capture molecules so as to provide a signal at the seconddetection location. A second product of the reaction binds to the thirdtype of capture molecule so as to provide a signal at the thirddetection location.

FIG. 21 shows a bottom view of the device of FIG. 20. From below,through the transparent additional carrier substrate 24, the layer 111of the non-transparent material arranged on top of the additionalcarrier substrate 24 can be seen. Groups of nine apertures are formed inthe layer of non-transparent material 111. Structurally, the layer ofnon-transparent material 111 comprises several apertures having a shapeto mask out any light other than the scattered light needed for themeasurement at the respective detection location. For optimalsuppression of the diffuse non-coherent background light at thedetection location, the diameter of a round aperture is chosen to belarger than the diameter do of the focal spot produced by the scatteredlight interfering at the detection location. In principle, the size isgiven by Abbe's formula for the calculation of the theoreticallypossible resolution of the microscope:d ₀=λ/2n _(s) sin α==λf/n _(s) Dwherein

-   λ is the vacuum wavelength of the coherent light propagating in the    planar waveguide,-   α is half the opening angle of the measurement zone,-   n_(s) is the refractive index of the substrate 3,-   f is the focal length of the measurement zone, and-   D is the diameter of the measurement zone.

Further apertures are formed in the non-transparent layers 111 in frontof and behind the apertures 21 (see FIG. 18) to determine an averagebackground signal. The shape of the apertures can be chosen so as tocorrespond to the shape of the focal spot formed by the light whichinterferes at the detection location. It may be advantageous to providean elongated aperture 21 (extending in the direction of propagation ofthe evanescent field) in order to avoid cutting off the light to bedetected in the detection location with the edge of the aperture, forexample in case of changes in the location of the focal spot caused bychanges in the refractive index of the sample applied to the outersurface of the planar waveguide or caused by small changes in thethickness of the planar waveguide.

While the embodiments of the invention have been described with the aidof the drawings, various modifications and changes to the describedembodiments are possible without departing from the general teachingunderlying the invention. Therefore, the invention is not to beunderstood as being limited to the described embodiments, but rather thescope of protection is defined by the claims.

The invention claimed is:
 1. A method for detection of bindingaffinities of target samples to binding sites, the method comprising thesteps of: providing a device comprising a planar waveguide arranged on asubstrate and an optical coupler, coupling coherent light of apredetermined wavelength into the planar waveguide such that thecoherent light propagates along the planar waveguide with an evanescentfield of the coherent light propagating along an outer surface of theplanar waveguide, attaching the target samples to the binding sitesarranged along a plurality of predetermined lines on the outer surfaceof the planar waveguide, detecting, at a predetermined detectionlocation, light of the evanescent field scattered by the target samplesbound to the binding sites arranged along the predetermined lines, thelight scattered by the target samples bound to the binding sites having,at the predetermined detection location, a difference in optical pathlength which is an integer multiple of the predetermined wavelength ofthe light, and from the light detected at the predetermined detectionlocation, determining the binding affinities of the target samples tothe binding sites.
 2. A method according to claim 1, wherein thepredetermined lines with the target samples attached thereto arearranged such that the coherent light scattered by the target samplesbound to the binding sites interferes at the predetermined detectionlocation with a difference in optical path length which is an integermultiple of the predetermined wavelength of the coherent light, so thata maximum of light scattered by the target samples bound to the bindingsites is located at the predetermined detection location, and whereinbinding of the target samples to the binding sites is determined whenthe said maximum of light scattered by the target samples bound to thebinding sites is detected at the predetermined detection location.
 3. Amethod according to claim 1, wherein the distance between adjacentpredetermined lines decreases in the direction of propagation of thelight of the evanescent field.
 4. A method according to claim 1, whereinthe plurality of predetermined lines along which the binding sites arearranged comprises curved lines, the curvature of the lines being suchthat light of the evanescent field scattered by the target samples boundto the binding sites interferes at a predetermined detection point asdetection location.
 5. A method according to according to claim 1,wherein the plurality of predetermined lines are arranged on the outersurface of the planar waveguide in a manner such that their locationsare geometrically defined by the equation$x_{j} = \frac{{\lambda\;{N\left( {A_{0} + j} \right)}} - \sqrt{{{n_{s}^{2}\left( {N^{2} - n_{s}^{2}} \right)}\left( {y_{j}^{2} + f^{2}} \right)} + {\left( {n_{s}\lambda} \right)^{2}\left( {A_{0} + j} \right)^{2}}}}{N^{2} - n_{s}^{2}}$wherein: λ is the vacuum wavelength of the propagating light, N is theeffective refractive index of the guided mode in the planar waveguide; Ndepends on the thickness and the refractive index of the planarwaveguide, the refractive index of the substrate, the refractive indexof a medium on the outer surface of the planar waveguide and thepolarization of the guided mode, n_(s) is the refractive index of thesubstrate, f is the thickness of the substrate, A₀ is an integer whichis chosen to be close to the product of the refractive index n_(s) andthe thickness f of the substrate divided by the wavelength λ, and j is arunning integer that indicates the index of the respective line.
 6. Amethod according to claim 1, wherein the binding sites comprise capturemolecules attached to the surface of the planar waveguide along thepredetermined lines only, the capture molecules being capable of bindingthe target samples.
 7. A method according to claim 1, wherein thebinding sites comprise capture molecules capable of binding the targetsamples, the capture molecules capable of binding the target samplesbeing arranged along the predetermined lines by dispensing capturemolecules capable of binding the target samples onto the outer surfaceof the planar waveguide and by deactivating those capture moleculeswhich are not arranged along the predetermined lines.
 8. A methodaccording to claim 1, wherein the planar waveguide has a refractiveindex (n_(w)) which is substantially higher than the refractive index(n_(s)) of the substrate and which is also substantially higher than therefractive index (n_(med)) of the medium on the outer surface of theplanar waveguide, such that for a predetermined wavelength of the lightthe evanescent field has a penetration depth in the range of 50 nm to200 nm.
 9. A method according to claim 1, wherein the device comprises afurther optical coupler for coupling out the light that has propagatedthrough the planar waveguide, wherein both the optical coupler forcoupling the light into the planar waveguide as well as the furtheroptical coupler for coupling out the light that has propagated throughthe planar waveguide comprise an optical grating for coherently couplinglight into and out of the planar waveguide.
 10. A method according toclaim 1, wherein the planar waveguide has a first end section and asecond end section which are arranged at opposite ends of the planarwaveguide with respect to the direction of propagation of the lightthrough the planar waveguide, the first end section and the second endsection each comprising a material which is absorptive at the wavelengthof the light propagating through the planar waveguide.
 11. A methodaccording to claim 1, wherein a plurality of measurement zones arearranged on the outer surface of the planar waveguide, wherein in eachmeasurement zone the binding sites are arranged along the plurality ofpredetermined lines.
 12. A method according to claim 11, wherein theplurality of measurement zones comprises measurement zones of differentsizes.
 13. A method according to claim 11, wherein each measurement zonehas an area larger than 25 μm², and wherein the plurality ofpredetermined lines has a distance between adjacent predetermined linesless than 1.5 μm.
 14. A method according to claim 11, wherein thebinding sites are arranged along at least two pluralities ofpredetermined lines in a single measurement zone, each of the twopluralities of predetermined lines being arranged such that the lightscattered by the target samples bound to the binding sites arrangedalong the respective plurality of predetermined lines interferes with adifference in optical path length which is an integer multiple of thepredetermined wavelength of the light at an individual detectionlocation for each plurality of predetermined lines with the individualdetection locations being spatially separated from each other.
 15. Amethod according to claim 11, wherein the device further comprises adiaphragm having an aperture which is arranged such that light at thedetection location is allowed to pass through the aperture while lightat a location different from the detection location is blocked by thediaphragm.
 16. A method according to claim 15, wherein the diaphragmfurther comprises at least one further aperture which is arrangedadjacent to the aperture when viewed in the direction of propagation ofthe light through the planar waveguide.
 17. A method according to claim11, wherein each measurement zone has an area larger than 25 μm², andwherein the plurality of predetermined lines has a distance betweenadjacent predetermined lines less than 1.0 μm.
 18. A method according toclaim 1, wherein the device further comprises a light source foremitting the coherent light of the predetermined wavelength, the lightsource being arranged such that the coherent light is coupled into theplanar waveguide via the optical coupler.
 19. A method according toclaim 1, wherein the device further comprises an optical imaging unit,the optical imaging unit being focused such as to produce an image ofthe detection location to an observation location.
 20. A methodaccording to claim 1, wherein the device further comprises aphoto-detector for measuring the intensity of the light at the detectionlocation.